5-lipoxygenase (5-LO) is the first committed enzyme in the pathway leading to leukotriene synthesis and is responsible for the conversion of arachidonic acid to LTA4 via the unstable intermediate 5-hydroperoxy eicosotetraenoic acid (Samuelsson et al., (1987) Science 237:1171; Samuelsson (1983) Science220:568). Leukotriene A4 can, in turn, be converted to leukotriene B4, C4, D4, or E4. Leukotrienes are potent local mediators which influence inflammatory and allergic response, including asthma, rheumatoid arthritis, psoriasis, thrombotic disease, ulcerative colitis, bronchitis, sinusitis, allergic and non-allergic rhinitis, and lupus. Leukotrienes (LTs) B4, C4, D4, and E4 have been shown experimentally to mimic the pathologic changes seen in asthma and to play a role in several inflammatory mechanisms that lead to airflow obstruction in asthma. Such mechanisms include bronchoconstriction, mucosal edema, increased secretion of mucus, and an inflammatory-cell infiltrate that is rich in eosinophils. (O""Bryne (1997) Chest 111:27S-34S). The slow-reacting substance of anaphylaxis is now known to be a mixture of leukotrienes C4, D4, and E4, all of which are potent bronchoconstrictors that exert their biological actions through specific ligand-receptor interactions thereby increasing vascular permeability and constricting smooth muscle (U.S. Pat. No. 5,750,565; Silverman, et al. (1998) Clin Exp Allergy 28 Suppl 5:164-70).
There have been research efforts to develop specific receptor antagonists or inhibitors of leukotriene biosynthesis, to prevent or minimize pathogenic inflammatory responses mediated by these compounds, including the inhibition of 5-LO. For example, European Patent Application Nos. 90117171.0 and 901170171.0 disclose indole, benzofuran, and benzothiophene lipoxygenase inhibiting compounds (U.S. Pat. No. 5,750,565). It has been established that treating patients with agents that have the capacity to inhibit 5-LO results in improvement in lung function, reduction in asthma symptoms, and decreased need for alternative asthma treatments (Persson et al., (1995) Anesthesiology 82:969; Israel et al., (1993) Ann. Int. Med. 119:1059). However, in patients with asthma, there is a heterogeneous response to treatment with 5-LO inhibitors. Mutations in the 5-LO gene sequence may be associated with responsiveness to therapy and/or susceptibility to diseases or disorders, e.g., 5 asthma.
The 5-LO gene has been cloned, both as a cDNA (Matsumoto et al., (1988) Proc. Natl. Acad. Sci. USA 85:3406; Dixon et al., (1988) Proc. Natl. Acad. Sci. USA 85:416; Balcarek et al., (1988) J. Biol. Chem. 263:13937) and as a genomic clone (Hoshiko et al., (1990) Proc. Natl. Acad. Sci. USA 87:9073; Funk et al., (1989) Proc. Natl. Acad. Sci. USA 86:2587). The 5-LO gene is approximately 85 kilobases in size and contains 14 exons and 15 introns. The region 88 to 212 base pairs upstream of the 5-LO translation start site contains a number of sequences known to be recognition sites for transcriptional regulators (Hoshiko et al., (1990) Proc. Natl. Acad. Sci. USA 87:9073, incorporated herein by reference). For example, binding sites for AP1 and Sp1, each of which can act as either a transcriptional activator or a transcriptional repressor, depending on context, are found in this region. With respect to the Sp1 binding site, it comprises 5 tandem Sp1 motifs (GGGCGG) found xe2x88x92147 to xe2x88x92176 base pairs upstream of the ATG translation start site. Deletions of the Sp1 motifs reduce transcription from this 5xe2x80x2 upstream regulatory element (Hoshiko et al. (1990) Proc. Natl. Acad. Sci. USA 87:9073). Sp1 binding sites comprising such motifs are similarly found in the promoter region of a variety of other genes (Li et al., (1995) Gene 164:229; Wariishi et al., (1995) Biochem. Biophys. Res. Commun. 216:729; Tang et al., (1995) Biochem. Biophys. Res. Commun. 213:673; Khachigian et al., (1995) J. Bio. Chem. 270:27679).
Certain polymorphisms in the 5-LO gene have been shown to be correlated with patient responsiveness to 5-LO inhibitor therapy (U.S. Pat. No. 6,090,547 by Drazen, et al. (2000)). Drazen et al. disclosed that asthma patients having polymorphisms associated with reduced 5-LO gene expression are less responsive to 5-LO inhibitor therapy than patients having normal 5-LO gene expression. Specifically, Drazen et al. showed that 5-LO promoters comprising a variant Sp1 binding site having 3, 4 or 6 Sp1 motifs were less active than the wild-type 5-LO promoter comprising a Sp1 bind site having 5 Sp1 motifs, and that asthma patients homozygous or heterozygous for 5-LO alleles comprising S-LO promoters having such variant Sp1 binding sites were less responsive to S-LO inhibitor therapy than patient homozygous for the wild-type 5-LO allele. Drazen et al. also showed that the 3 Sp1 motif binding site is in linkage disequilibrium with a G to A polymorphism in exon 2 of the 5-LO gene, and that the 4 Sp1motif binding site is in linkage disequilibrium with a C to T polymorphism in exon 1 of the 5-LO gene.
It would also be desirable to identify additional polymorphisms within the 5-LO gene which are in linkage disequilibrium with a variant Sp1 binding site in the 5-LO promoter. It would further be desirable, to provide prognostic, diagnostic, pharmacogenomic and therapeutic methods utilizing the identified polymorphisms.
The present invention relates to polymorphisms in the 5-LO gene. The invention is based, in part, on the discovery of a novel 5-LO haplotype which comprises five sequence polymorphisms within the 5-LO gene that are in linkage disequilibrium with each other. The five 5-LO polymorphisms are set forth in Table 1. Three of these, 5loprr1, 5lo01a and 5lo04a, are newly identified polymorphisms. The other two, 5lonrra and 5lonrrb, have been described by In et al. in J. Clinical invest. 99:1130-1137 (1997). The present invention is based, also in part, on the discovery that the haplotype of the invention is in linkage disequilibrium with any one of three variant Sp1 binding sites in the 5-LO promoter region that were previously reported by Drazen et al. U.S. Pat. No. 6,090,547). Accordingly, any one of the five polymorphisms of the invention is a marker for the other four polymorphisms and a variant Sp1 binding site having 3, 4 or 6 Sp1 motifs in the 5-LO promoter region. Moreover, since Drazen et al. (id.) demonstrated that asthma patients homozygous or heterozygous for a 5-LO allele comprising any one of such variant Sp1 binding sites are less susceptible to effective treatment with 5-LO inhibitor therapy, the haplotype of the invention is also a marker of reduced responsiveness amongst inflammatory or allergy disease patients to 5-LO inhibitor therapy.
The present invention is based further, in part, on the discovery that the 5lo01a polymorphism of the invention is associated with an abnormally low eosinophil count. That is, individuals heterozygous or homozygous for the 5lo01a polymorphism have been found to have significantly lower eosinophil levels compared to individuals not having the polymorphism. Thus, the haplotype of the invention and each of its component polymorphisms are all markers of low eosinophil levels.
The invention provides a method for identifying an inflammatory or allergy disease patient who is less susceptible to effective treatment with a 5-LO inhibitor, comprising determining the presence or absence of a 5-LO allelic variant comprising a sequence selected from the group consisting of those set forth in SEQ ID NO: 4, SEQ ID NO:5, and SEQ ID NO:6, or the complement of the sequence in a nucleic acid sample from an inflammatory or allergy disease patient, wherein the presence of the 5-LO allelic variant in the sample indicates that the patient is less susceptible to effective treatment with a 5-LO inhibitor. In one embodiment, the 5-LO allelic variant comprises the sequences of those set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or the complement of each of those sequences. In another embodiment, the 5-LO allelic variant comprises the sequences of those set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, or the complement of each of those sequences. In yet another embodiment, the 5-LO allelic variant comprises: (a) a sequence selected from the group consisting of those set forth in SEQ ID NO: 4, SEQ ID NO:5, and SEQ ID NO:6, or the complement of each of those sequences; and (b) a variant Sp1 binding site having a 3, 4 or 6 Sp1 motif. In yet another embodiment, the 5-LO allelic variant comprises: (a) a sequence selected from the group consisting of those set forth in SEQ ID NO: 4, SEQ ID NO:5, and SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, or the complement of each of those sequences; and (b) a variant Sp1 binding site having a 3, 4 or 6 Sp1 motif.
The invention also provides a method for identifying a patient with an abnormally low eosinophil level, comprising determining the presence or absence of a 5-LO allelic variant comprising a sequence selected from the group consisting of those set forth in SEQ ID NO: 4, SEQ ID NO:5, and SEQ ID NO:6, or the complement of the sequence in a nucleic acid sample from a patient, wherein the presence of the 5-LO allelic variant in the sample indicates that the patient has an abnormally low eosinophil level. In one embodiment, the 5-LO allelic variant comprises the sequences of those set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or the complement of each of those sequences. In another embodiment, the 5-LO allelic variant comprises the sequences of those set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, or the complement of each of those sequences. In yet another embodiment, the 5-LO allelic variant comprises: (a) a sequence selected from the group consisting of those set forth in SEQ ID NO: 4, SEQ ID NO:5, and SEQ ID NO:6, or the complement of each of those sequences; and (b) a variant Sp1 binding site having a 3, 4 or 6 Sp1 motif In yet another embodiment, the 5-LO allelic variant comprises: (a) a sequence selected from the group consisting of those set forth in SEQ ID NO: 4, SEQ ID NO:5, and SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, or the complement of each of those sequences; and (b) a variant Sp1 binding site having a 3, 4 or 6 Sp1 motif. In a preferred embodiment, the patient is an inflammatory or allergy disease patient. In a more preferred embodiment, the patient is an asthma patient.
It is believed that inflammatory or allergy disease patients having abnormally low eosinophil levels have milder disease symptoms than patients having normal eosinophil levels. Accordingly, the instant invention further provides a method for determining the severity of disease symptoms in an inflammatory or allergy disease patient comprising determining the presence or absence of a 5-LO allelic variant comprising a sequence selected from the group consisting of those set forth in SEQ ID NO: 4, SEQ ID NO:5, and SEQ ID NO:6, or the complement of the sequence in a nucleic acid sample from the patient, wherein the presence of the 5-LO allelic variant in the sample indicates that the patient has or will less severe disease symptoms than patients having wild-type 5-LO alleles. In one embodiment, the 5-LO allelic variant comprises the sequences of those set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or the complement of each of those sequences. In another embodiment, the 5-LO allelic variant comprises the sequences of those set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, or the complement of each of those sequences. In yet another embodiment, the 5-LO allelic variant comprises: (a) a sequence selected from the group consisting of those set forth in SEQ ID NO: 4, SEQ ID NO:5, and SEQ ID NO:6, or the complement of each of those sequences; and (b) a variant Sp1 binding site having a 3, 4 or 6 Sp1 motif. In yet another embodiment, the 5-LO allelic variant comprises: (a) a sequence selected from the group consisting of those set forth in SEQ ID NO: 4, SEQ ID NO:5, and SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, or the complement of each of those sequences; and (b) a variant Sp1 binding site having a 3, 4 or 6 Sp1 motif. In a preferred embodiment, the patient is an asthma patient.
The invention also provides isolated nucleic acids comprising the novel 5-LO polymorphisms and the haplotype of the invention. The nucleic acid molecules of the invention include specific 5-LO allelic variants which differ from the reference 5-LO sequences set forth in SEQ ID NO:1 (GI 187166), SEQ ID NO: 2 (GI 8247778), SEQ ID NO:3 (GI 4502056). The preferred nucleic acid molecules of the invention comprise newly identified 5-LO allelic variants or portions thereof having the any of the novel polymorphisms shown in Table 1 (i.e., those comprising the sequence of any of those set forth in SEQ ID NOs:4-6 or the complement thereof), polymorphisms in linkage disequilibrium with the polymorphisms shown in Table 1, and combinations thereof. The haplotype also includes known polymorphisms. These known polymorphisms include a guanine to adenine change in the 5xe2x80x2 upstream regulatory element at residue 84 of SEQ ID NO:1 (GI 187166) and a guanine to adenine change in the 5xe2x80x2 upstream regulatory element at residue 137 of SEQ ID NO:1 (GI 187166) (In, et al. (1997) J. Clinical Invest. 99:1130-1137). These known polymorphisms are also listed in Table 1 under xe2x80x9cKnown SNPsxe2x80x9d and correspond to SEQ ID NOs.:7 and 8. These known polymorphisms may be used in combination with the newly identified polymorphisms of the present invention in the diagnostic, prognostic, pharmacogenomic and therapeutic methods described herein.
The haplotype of the invention is in linkage disequilibrium with known polymorphisms in the promoter region of the 5-LO gene. These polymorphisms are variations at the Sp1 binding site as described in U.S. Pat. No. 6,090,547 and In, et al. ((1997) J. Clin. Invest. 99(5):1130-1137). The wild-type 5-LO gene has a Sp1 binding site comprising five Sp1 motifs in tandem. Each of the Sp1 motifs consists of the nucleotide sequence xe2x80x9cGGCGGGxe2x80x9d (SEQ ID NO:61). The wild-type Sp1 site is located at nucleotide residues 1670-1699 in the reference sequence GI 187166 (SEQ ID NO:1). Polymorphisms contained within the Sp1 binding site repeat which are referred to herein as xe2x80x9cvariant Sp1 binding sitesxe2x80x9d have the deletion of one, the deletion of two, or the addition of one, Sp1 binding motif in the promoter region of the 5-LO gene resulting in three, four, or six Sp1 motifs.
The nucleic acid molecules of the invention can be double- or single-stranded. Accordingly, in one embodiment of the invention, a complement of the nucleotide sequence is provided wherein the polymorphism has been identified. For example, where there has been a single nucleotide change from a guanine to an adenine in a single strand, the complement of that strand will contain a change from cytidine to thymine at the corresponding nucleotide residue.
Nucleic acids of the invention can function as probes or primers, e.g., in methods for determining the allelic identity of a 5-LO polymorphic region in a nucleic acid of interest. The invention further provides vectors comprising the nucleic acid molecules of the present invention; host cells transfected with said vectors whether prokaryotic or eukaryotic; and transgenic non-human animals which contain a heterologous form of a functional or non-functional 5-LO allele described herein. Such a transgenic animal can serve as an animal model for studying the effect of specific 5-LO allelic variations, including mutations, as well as for use in drug screening and/or recombinant protein production.
The invention further provides methods for determining the molecular structure of at least a portion of a 5-LO gene. In a preferred embodiment, the method comprises contacting a sample nucleic acid comprising a 5-LO gene sequence with a probe or primer having a sequence which is complementary to a 5-LO gene sequence, carrying out a reaction that would amplify and/or detect differences in a region of interest within the 5-LO gene sequence, and comparing the result of each reaction with that of a reaction with a control (known) 5-LO gene (e.g., a 5-LO gene from a human not afflicted with an inflammatory condition e.g., asthma, or another disease associated with an aberrant 5-LO activity) so as to determine the molecular structure of the 5-LO gene sequence in the sample nucleic acid. The method of the invention can be used for example in determining the molecular structure of at least a portion of an exon, an intron, a 5xe2x80x2 upstream regulatory element, or the 3xe2x80x2 untranslated region. In a preferred embodiment, the method comprises determining the identity of at least one nucleotide. In another preferred embodiment, the method comprises determining the nucleotide at residue 1000 of the reference sequence GI 8247778, any one of residues 472-477 of the reference sequence GI 187166, and/or residue 559 of the reference sequence GI 187166, and combinations thereof. In another embodiment, the method comprises determining the nucleotide at residue 1000 of the reference sequence GI 8247778, any one of residues 472-477 of the reference sequence GI 187166, and/or residue 559 of the reference sequence GI 187166, the nucleotide at residue 84 of GI 187166, and/or the nucleotide at residue 137 of GI 187166. In a further embodiment, the method comprises determining the nucleotide at residue 1000 of the reference sequence GI 8247778, any one of residues 472-477 of the reference sequence GI 187166, and/or residue 559 of the reference sequence GI 187166, and the number of Sp1 motifs in the promoter region of the 5-LO gene.
In another preferred embodiment, the method comprises determining the nucleotide content of at least a portion of a 5-LO gene, such as by sequence analysis. In yet another embodiment, determining the molecular structure of at least a portion of a 5-LO gene is carried out by single-stranded conformation polymorphism (SSCP). In yet another embodiment, the method is an oligonucleotide ligation assay (OLA). Other methods within the scope of the invention for determining the molecular structure of at least a portion of a 5-LO gene include hybridization of allele-specific oligonucleotides, sequence specific amplification, primer specific extension, and denaturing high performance liquid chromatography (DHPLC). In at least some of the methods of the invention, the probe or primer is allele specific. Preferred probes or primers are single stranded nucleic acids, which optionally are labeled.
The invention further provides forensic methods based on detection of polymorphisms in the 5-LO gene.
The invention also provides probes and primers comprising oligonucleotides which hybridizes to at least 6 consecutive nucleotides of any of the sequences set forth in SEQ ID NOs: 4-6, or to the complement of any of such sequences, or naturally occurring mutants or variants thereof. In preferred embodiments, the probe/primer further includes a label attached thereto, which is capable of being detected.
In another embodiment, the invention provides a kit for amplifying and/or for determining the molecular structure of at least a portion of a 5-LO gene, comprising a probe or primer capable of hybridizing to a 5-LO gene and instructions for use. In one embodiment, the probe or primer is capable of hybridizing to a 5-LO intron. In another embodiment, the probe or primer is capable of hybridizing to a 5-LO allelic variant, preferrably a variant corresponding to 5 loprr1, 5lo01a or 5lo04a. In another preferred embodiment, the polymorphic region is located in the 5xe2x80x2 upstream regulatory element. In a preferred embodiment, determining the molecular structure of a region of a 5-LO gene comprises determining the identity of the allelic variant of the polymorphic region. Determining the molecular structure of at least a portion of a 5-LO gene can comprise determining the identity of at least one nucleotide or determining the nucleotide composition, e.g., the nucleotide sequence.
A kit of the invention can be used, e.g., for determining whether a patient will or will not be responsive to effective treatment of a disease associated with a specific 5-LO allelic variant, e.g., asthma, with a 5-LO inhibitor. In a preferred embodiment, the invention provides a kit for determining whether a patient will or will not be responsive to treatment of a inflammatory or allergic disease or condition associated with abnormal leukotriene synthesis, for example, asthma. The kit of the invention can also be used in selecting the appropriate drug to administer to a patient to treat such a disease or condition.
In another aspect, the invention provides a kit for determining whether a inflammatory or allergic disease patient has a more moderate or more severe disease phenotype associated with a specific 5-LO allelic variant of a polymorphic region. In one embodiment, the disease or disorder is characterized by an abnormal 5-LO activity, e.g., aberrant 5-LO expression. In another embodiment, the disease or disorder is characterized by an abnormal eosinophil levels.
Other features and advantages of the invention will be apparent from the following detailed description and claims.